Monolith separation medium for chromatographic use and method of producing the same

ABSTRACT

A monolith separation medium comprising a skeletal phase and continuous pores forming a three-dimensional network structure, which has a functional group enabling the introduction of a new functional group on the surface of the skeletal phase. The skeletal phase has an average diameter of a submicron to micrometer size and is in a co-continuous structure of the non-particle-aggregation type. It is composed of an addition polymer of 1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane as an epoxy compound with a bifunctional or higher amine compound, is rich in organic matters and is free from any aromatic-origin carbon atom. Thus, it is an organic polymer monolith separation medium of the non-particle-aggregation type.

TECHNICAL FIELD

The present invention relates to a monolith separation medium forchromatographic use which comprises a skeletal phase composed of anorganic polymer, which has a three-dimensional network structure,interconnected pores, and a method of producing the same.

BACKGROUND ART

It is known that porous bodies for chromatographic use having boththrough-flow channels and a skeleton (referred to as “monoliths”) haveadvantages which cannot be obtained by conventional particle-packedcolumns. For example, when such a porous body is used as a separationmedium for HPLC, high column performance can be achieved due to its wideflow channels and thin skeleton and performance degradation can bereduced even at a high flow rate due to its low pressure loss.High-performance monoliths mainly made of silica gel have already beenrealized by microstructure control, and some of them are commerciallyavailable.

On the other hand, organic polymer monoliths are also known.Conventional organic polymer monoliths are formed by combinations ofhydrophobic monomer components and poor solvents as pore-formingsolvents (porogens). In a solution system containing a hydrophobicmonomer component and a poor solvent, Van der Waals force betweengrowing polymer chains becomes stronger than the steric hindrance of thepolymer chains, and therefore the polymer chains aggregate. This causesnuclear formation due to the entanglement of the polymer chains, growthof microgel particles due to the aggregation of the polymer chains, anda rapid increase in the surface energy of the system. Further, themicrogel particles aggregate and become coarse by similar growth(fractal growth) so that a gel is formed. In the case of such a solutionsystem, gel formation is due to particle aggregation, and phaseseparation competes with gelation and occurs at a very early stage sothat a monolithic structure with a small specific surface area is fixed.In this case, a particle-aggregation-type monolith is formed. Therefore,the conventional organic polymer monoliths do not have a skeletalstructure and have a large pore tortuosity factor, and thus inherentlyhave problems such as an increase in back pressure at a high flow rateand morphological change due to their compressibility.

Further, the conventional organic polymer monoliths also have a problemthat when directly formed in empty column tubes with an inner diameterof 1 mm or more, they are peeled off from the column tubes due to theirown compressibility. This makes it difficult to directly form organicpolymer monoliths in empty column tubes.

In order to solve such problems, the present inventors have extensivelystudied, and as a result have developed a monolith separation mediumwhose skeletal phase is composed of an addition polymer of a bi- orhigher-functional epoxy compound and a bi- or higher-functional aminecompound, and has a functional group allowing a new functional group tobe introduced thereinto (see Patent Document 1).

Further, another organic polymer monolith is also known, which has askeletal phase composed of a copolymer of GMA (glycidyl methacrylate)and EDMA (ethylene dimethacrylate) and having amino groups introduced asfunctional groups by reacting epoxy groups present on the surfacethereof with ethylenediamine (see Non-Patent Documents 1 and 2).

Patent Document 1: WO2007/083348 A1

Non-Patent Document 1: Journal of Peptide Science, Vol. 10, pp. 719-730,2004

Non-Patent Document 2: Journal of Combinational Chemistry, Vol. 4, no.1, pp. 33-37, 2002

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

It is an object of the present invention to provide a monolithseparation medium whose skeletal phase is composed of an additionpolymer of a bi- or higher-functional epoxy compound and a bi- orhigher-functional amine compound, that is, a monolith separation mediumhaving a skeletal phase which is similar to that of the monolithseparation medium disclosed in Patent Document 1 and which is formed bya novel material combination.

Means for Solving the Problem

In order to achieve the above object, the present inventors have foundthat a porous body having a very uniform skeletal structure can beobtained by dissolving an epoxy compound having a specific molecularstructure in a porogen, adding a bi- or higher-functional amine compoundthereto, heating the solution to obtain a polymer, spinodallydecomposing the porogen and the polymer, stably cross-linking thepolymer before a non-particle-aggregation-type co-continuous structureof the polymer and the porogen transits to a particle aggregationstructure due to the growth of phase separation to freeze theco-continuous structure, and removing the porogen. The epoxy compoundhaving a specific molecular structure is1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane which is a liquid epoxycompound having four epoxy groups and which undergoes polymerization notat room temperature but at elevated temperatures.

The thus obtained organic polymer monolith separation medium accordingto the present invention comprises a skeletal phase, interconnectedpores formed by a three-dimensional network of the skeletal phase, and afunctional group present on the skeletal phase to allow a new functionalgroup to be introduced into the skeletal phase, wherein the skeletalphase has an average diameter in submicron to micrometer size range anda non-particle-aggregation-type co-continuous structure, is composed ofan addition polymer of 1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane asan epoxy compound and a bi- or higher-functional amine compound, is richin organic matter, and contains no aromatic-origin carbon atom orheterocyclic ring.

The term “functional group present on the skeletal phase to allow a newfunctional group to be introduced into the skeletal phase” includes ahydroxyl group generated by the reaction between an epoxy group and anamino group as well as remaining unreacted amino and epoxy groups.

The present invention also provides a method for producing the organicpolymer monolith separation medium according to the present invention,the method comprising the steps of:

(A) heating 1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane as an epoxycompound and a bi- or higher-functional amine compound in a porogen at atemperature in a range of 60 to 200° C. to polymerize the epoxy compoundwith the amine compound to obtain a gel; and

(B) washing the gel with a solvent such as water to remove the porogenso that a skeletal phase remains.

The temperature of a polymerization reaction between the epoxy compoundand the amine compound dissolved in the porogen is not particularlylimited as long as it is suitable for the polymerization reaction toproceed, and is appropriately set depending on the kinds of epoxycompound, amine compound, and porogen to be used.

The amine compound is used as a curing agent. As the amine compound, apolyamine which is selected from the group consisting of aliphaticamines, alicyclic polyamines, and aliphatic polyamide amines, and whichcontains two or more primary amines can be used.

Examples of the aliphatic amines include ethylenediamine,diethylenetriamine, triethylenetetramine, tetraethylenepentamine,iminobispropylamine, bis(hexamethylene)triamine,1,3,6-trisaminomethylhexane, polymethylenediamine,trimethylhexamethylenediamine, and polyetherdiamine and the like.

Examples of the alicyclic polyamines include isophoronediamine,menthanediamine, N-aminoethylpiperazine, 3,9-bis(3-aminopropyl)2,4,8,10-tetraoxaspiron, bis(4-aminocyclohexyl)methane, and modifiedproducts thereof.

Examples of the aliphatic polyamide amines include those formed frompolyamines and dimer acids.

Particularly preferred examples of the amine compound includebis(4-aminocyclohexyl)methane andbis(4-amino-3-methylcyclohexyl)methane.

The porogen refers to a solvent which can dissolve the epoxy compoundand the curing agent as well as can cause reaction-induced phaseseparation after polymerization of the epoxy compound with the curingagent. Examples of such a porogen include cellosolves, esters, andglycols.

Examples of the cellosolves include methyl cellosolve and ethylcellosolve.

Examples of the esters include ethylene glycol monomethyl ether acetateand propylene glycol monomethyl ether acetate and the like.

Examples of the glycols include polyethylene glycol and polypropyleneglycol and the like.

Among these porogens, polyethylene glycols having a molecular weight of600 or less are preferred, and polyethylene glycols having a molecularweight of 300 or less are particularly preferred.

According to the production method of the present invention, a suitablemolar ratio between the epoxy compound and the amine compound used asraw materials is in the range of 1:1 to 1:3 (epoxy compound: aminecompound).

A suitable amount of the porogen added is 1 to 99 wt % with respect tothe total weight of the epoxy compound, the amine compound, and theporogen.

1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane used in the presentinvention is a chiral compound having optical enantiomers, and may beeither in racemic or optically active form.

The amine compound may also be a chiral compound. In this case, theamine compound may be either in racemic or optically active form.

An optical resolution separation medium for liquid chromatographic useintended to separate a racemic mixture into its S- and R-enantiomers canbe realized when both the epoxy compound and the amine compound areoptically active.

EFFECTS OF THE INVENTION

According to the present invention, a cross-linking reaction occursbefore particle aggregation occurs, which makes it possible to realizean organic polymer monolith having a clear skeletal phase and aco-continuous structure. Further, the cross-linking reaction easilyoccurs due to the use of a polyfunctional epoxy compound, and thereforeit is expected that the shrinkage of the organic polymer monolith due tothe cross-linking reaction will be reduced. Particularly,1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane as an epoxy compound isliquid and poorly reactive at room temperature, and therefore can bemore easily injected into a column or the like as compared to an epoxycompound which needs to be dissolved in a porogen at an elevatedtemperature, such as a solid epoxy compound. Further,1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane is a tetrafunctionalepoxy compound, which makes it possible to reduce shrinkage duringcross-linking and gelation which occur after phase separation.

As described above, the monolith separation medium according to thepresent invention has a skeletal phase which has an average diameter insubmicron to micrometer size range and a non-particle-aggregation-typeco-continuous structure, and which is composed of an addition polymer ofa bi- or higher-functional epoxy compound and a bi- or higher-functionalamine compound, and which is rich in organic matter, and which containsno aromatic-origin carbon atom, and is therefore useful as a stationaryphase for liquid chromatographic use and achieves high performance whichcannot be achieved by conventional monolith separation media at all.Further, the monolith separation medium according to the presentinvention can be used in various-sized columns ranging from capillarycolumns to general-purpose columns.

According to the production method of the present invention, a monolithseparation medium can be very easily produced by heating an epoxycompound and an amine compound in a porogen to polymerize the epoxycompound with the amine compound. Therefore, the production methodaccording to the present invention can be effectively used to producevarious-sized columns ranging from capillary columns to columns having arelatively large diameter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scanning electron microscope image of an organic polymermonolith capillary column produced in Example 1.

FIG. 2 is a chromatogram showing separation of uracil and benzene usingthe capillary column shown in FIG. 1.

FIG. 3 is a chromatogram showing separation of uracil and benzene usingan organic polymer monolith capillary column produced in ComparativeExample.

DETAILED DESCRIPTION OF THE INVENTION

A separation medium according to the present invention is ahigh-performance and non-particle-aggregation-type polymer monolithseparation medium formed by a specific material combination of1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane as an epoxy compound anda curing agent. More specifically, the epoxy compound, that is,1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane is a tetrafunctionalnon-aromatic epoxy compound, and the curing agent is a bi- orhigher-functional non-aromatic amine compound. Such a materialcombination makes it possible to obtain a high-performance andnon-particle-aggregation-type polymer monolith separation medium havinga three-dimensional branching structure.

Hereinbelow, an epoxy compound and a curing agent which can be used inthe present invention will be exemplified.

EXAMPLES Example 1

A separation medium according to the present invention and a methodsuitable for producing the same will be described with reference toExample 1. In Example 1, 1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane(BDAC) was used as an epoxy compound, bis(4-aminocyclohexyl) methane(BACM) was used as an amino compound, and polyethylene glycol with amolecular weight of 300 (manufactured by Nacalai Tesque Inc. under thetrade name of “PEG300”) was used as a porogen. The1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane may be either in racemicor optically active form. The BDAC and BACM have the following chemicalstructural formulas.

(Production of Polymer Monolith)

0.93 g of the BDAC, 0.47 g of the BACM, and 3.6 g of the PEG300 wereprepared, injected into a fused quartz capillary tube at roomtemperature, and heated in an oil bath at 150° C. for 24 hours topolymerize the BDAC with the BACM. After the completion of thepolymerization, an obtained gel was washed with methanol and water.

FIG. 1 is a scanning electron micrograph of a capillary column filledwith an organic polymer monolith produced by polymerization inExample 1. As can be seen from FIG. 1, the organic polymer monolith hasa skeletal phase having a micrometer-sized average diameter and anon-particle-aggregation-type co-continuous structure and pores formedby a three-dimensional network structure of the skeletal phase.

Separation of uracil and benzene was performed using the organic polymermonolith capillary column (inner diameter: 100 μm, length: 20 cm)produced in Example 1. FIG. 2 is a chromatogram showing separation ofuracil and benzene. As a mobile phase, a 60% aqueous acetonitrilesolution (pH 7.0 adjusted by 20 mM phosphate buffer) was used. Thetemperature of the capillary column was room temperature. Detection wascarried out by measuring ultraviolet absorption at 210 nm. In FIG. 2,the former peak is assigned to uracil, the latter peak is assigned tobenzene, and N represents the number of theoretical plates.

The number of theoretical plates was calculated by the half-height widthmethod using the following formula:

N=5.54(t _(r) /W _(0.5h))²

where t_(r) represents retention time and W_(0.5h) represents a peakwidth at half-height.

Comparative Example

As an epoxy compound,2-[(4-{1-methyl-1-[4-(2oxiranylmethoxy)phenyl]ethyl}phenoxy)methyl]oxirane (BADE) being an aromatic compound was used. As an aminecompound, BACM, which was the same as that used in Example 1, was used.As a porogen, polyethylene glycol with a molecular weight of 200(manufactured by Nacalai Tesque Inc. under the trade name of “PEG200”)was used.

0.52 g of the BACM was melted in 7.20 g of the PEG200 by heating, andthen 2.33 g of the BADE was added thereto and mixed by stirring. Thethus obtained solution was injected into a fused quartz capillary tubeand heated in an oven at 120° C. for 1 hour to polymerize the BADE withthe BACM.

After the completion of the polymerization, an obtained gel was washedwith water and methanol and then vacuum-dried.

Separation of uracil and benzene was performed using an organic polymermonolith capillary column (inner diameter: 100 μm, length: 20 cm)produced in Comparative Example. FIG. 3 is a chromatogram showingseparation of uracil and benzene. As a mobile phase, a 60% aqueousacetonitrile solution (pH 7.0 adjusted by 20 mM phosphate buffer) wasused. The temperature of the capillary column was room temperature, theflow rate of the mobile phase was 0.15 mL/min, the linear velocity was1.01 mm/sec, and the pressure was 50 kg/cm². Detection was carried outby measuring ultraviolet absorption at 210 nm in a capillary tube withan inner diameter of 50 μm placed 9 cm away from the column.

In FIG. 3, the former peak is assigned to uracil and the latter peak isassigned to benzene. The number of theoretical plates for uracil andbenzene calculated by the half-height width method were 3085 and 378,respectively, which were lower than those of the organic polymermonolith capillary column according to the present invention produced inExample 1. The reason for this can be considered as follows: the epoxycompound used in Comparative Example is an aromatic compound, andtherefore, a formed skeletal phase contains aromatic-origin carbonatoms.

INDUSTRIAL APPLICABILITY

The monolith separation medium according to the present invention can beused as a stationary phase in various-sized liquid chromatographiccolumns ranging from capillary columns to general-purpose columns.

1. An organic polymer gel monolith separation medium for chromatographicuse comprising: a skeletal phase; interconnected pores formed by athree-dimensional network of the skeletal phase; and a functional grouppresent on the skeletal phase to allow a new functional group to beintroduced into the skeletal phase, wherein the skeletal phase has anaverage diameter in submicron to micrometer size range and anon-particle-aggregation-type co-continuous structure, is composed of anaddition polymer of 1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane as anepoxy compound and a bi- or higher-functional amine compound, is rich inorganic matter, and contains no aromatic-origin carbon atom orheterocyclic ring.
 2. The monolith separation medium according to claim1, wherein the amine compound is a polyamine, which is selected from thegroup consisting of aliphatic amines, alicyclic polyamines, andaliphatic polyamide amines, and which contains two or more primaryamines.
 3. The monolith separation medium according to claim 2, whereinthe amine compound is bis(4-aminocyclohexyl)methane as an alicyclicpolyamine.
 4. The monolith separation medium according to claim 1,wherein the epoxy compound and the amine compound are both opticallyactive to allow optical enantiomers to be separated from each other. 5.A method of producing the monolith separation medium according to claim1, comprising the steps of: (A) heating1,3-bis(N,N′-diglycidylaminomethyl)cyclohexane as an epoxy compound anda bi- or higher-functional amine compound in a porogen at a temperaturein a range of 60 to 200° C. to polymerize the epoxy compound with theamine compound to obtain a gel; and (B) washing the gel with a solventto remove the porogen so that a skeletal phase remains.
 6. Theproduction method according to claim 5, wherein the amine compound is analiphatic amine, an alicyclic polyamine, or an aliphatic polyamideamine.
 7. The production method according to claim 6, wherein the aminecompound is bis(4-aminocyclohexyl)methane as an alicyclic polyamine. 8.The production method according to claim 5, wherein the porogen is acellosolve, an ester, or a glycol.
 9. The production method according toclaim 8, wherein the porogen is polyethylene glycol.